Ultrasonication

The DNA libraries were prepared using 500 ng of genomic DNA, starting with ultrasonication (Covaris, Woburn, MA, USA) to produce double-strand DNA fragments with an average length of 280 bp. End-Repair and A-tailing were then applied to facilitate ligation of the adapters, containing unique barcodes for each sample, specific to the Illumina technology for amplification and sequencing. KAPA Hyper Prep kit was used for these steps, according to the manufacturer’s instructions.

The DNA libraries were prepared using 500 ng of genomic DNA (extracted from frozen tissue), starting with ultra-sonication (Covaris) to produce double-strand DNA fragments of approximately 280 bp. End-Repair and A-tailing were applied to facilitate ligation of the adapters, containing unique barcodes for each sample, specific to the Illumina technology for amplification and sequencing. KAPA Hyper Prep kit was used, according to the manufacturer’s instructions.

Next-generation sequencing libraries were prepared as described previously (Kanoh et al, 2015 (link)). The input and the immunoprecipitated DNAs were fragmented to an average size of ∼150 bp by ultra-sonication (Covaris). The fragmented DNAs were end-repaired, ligated to sequencing adapters, and amplified using NEBNext Ultra II DNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (New England Biolabs). The amplified DNA (around 275 bp in size) was sequenced on Illumina MiSeq to generate single reads of 100 bp. The generated ChIP or input sequences were aligned to the S. pombe genomic reference sequence provided from PomBase by Bowtie 1.0.0 using default settings. Peaks were called with model-based analysis of ChIP-Seq (MACS2.0.10) using the following parameters: macs2 callpeak -t ChIP.sam -c Input.sam -f SAM -g 1.4e10⁷ -n result_file –B -q 0.01. The pileup graphs were loaded on Affymetrix Integrated Genome Browser (IGB 8.0). To identify consensus conserved sequences for Rif1 binding, 300-bp DNA segments around the summits of the 128 or 169 Rif1bs identified by MACS2 were extracted and analyzed by MEME suite (Bailey et al, 2015 (link)).

Genomic DNA was isolated as before. DNA was fragmented with Covaris ultrasonication with default settings for the generation of fragments of 350 basepair average size. Libraries were constructed with the TruSeq DNA PCR-Free Kit (Illumina 20015962). Libraries were quantified with the KAPA Illumina Quantification Kit (KAPA KK4824).